Progress 10/01/12 to 09/30/16
Outputs
Target Audience:Animals scientist, veterinarians, graduate students, bull stud managers, cattle ranchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In last five years, two graudate students, one resrach technician andtwo postdocs contributed to the descibed studeis.Two full professors and one research associate professor co-developed the genomic project described above and jointly applied for funding from NIH-USDA Dual Purpose Dual Benefit program, ranked in top 3% of all applications evaluated in February 2016. If funded, this project will provide ample opportunites for professional developemnt of postdocs, research faculty and graduate students. How have the results been disseminated to communities of interest?In addition to publications and video presentations reported on previous pages, following presentations made in 2014-2016 included data from this project: 2016: 11th International Scientific Conference on Biotechnology and Quality of Raw Materials and Foodstuffs, January 27-29, 2016, Stará Lesná, Slovak Republic. 2015: Warnick Lecturer, D.H. Barron Reproductive and Perinatal Biology Research Program, University of Florida. November 3, 2015 2015: Gordon Research Conference on "Nanoscale Science and Engineering for Agriculture and Food Systems", June 7-12, 2015, Bentley University, Waltham, MA 2015: XIX Curso "Novos Enfoques na Producao e Reproducao de Bovinos", XIX Course on New Advanced in Bovine Production & Reproduction, Uberlandia, Brazil, March 19 & 20, 2015 2014: IV International Scientific-Technical Day on Bovine Reproduction, Congress Palace, Huesca, Spain (delivered via videopresentation, scheduling conflict prevented travel to Spain) 2014: Joint Closing & Opening Session, World Congress of Andrology and Applied Animal Andrology Symposium, Newcastle, NSW, AUSTRALIA, August 8-15, 2014 2014: USDA Agricultural Outlook Forum, February 20-21, 2014, Crystal City, Virginia (pre-recorded presentation), What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our goal isto identify the loss of function (LOF) polymorphisms that influence spermatogenesis and sperm function during fertilization. Since LOF mutations are presumably deleterious and at low frequency in the population, we would expect much higher frequencies of heterozygous than homozygous bulls. To avoid the high false negative rate in the detection of heterozygotes that is due to their lower average individual allele read depths, we usually sequence individuals to a target depth of ~30X to enable the accurate identification of rare, exclusively heterozygous alleles. By comparison to genotypes produced for these bulls using the Illumina BovineHD Beadchip (assays 778K loci), we concluded that our polymorphism discovery pipeline is as accurate as the commercial genotyping platforms. In the analysis of the 219 sequenced bulls, we predicted 3,601 high confidence (polymorphism present in at least two individuals) genes with LOF polymorphisms or SNPs causing predicted non-tolerated, non-synonymous amino acid changes. Of these genes, 29 are annotated as being directly associated with spermatogenesis/spermand 281 are known to be essential for life based upon orthologous mouse knockouts. In the essential-for-life group, are genes important for fertilization and early development, ribosomal biogenesis and embryo protein-turnover. Deleterious LOF alleles were also found in eight pregnancy associated glycoprotein genes important for pregnancy establishment and maintenance [50].Five of the sperm-related LOF geneshave male infertile/subfertile KOs in mice and polymorphisms in at least two of the gene orthologs are associated with human male infertility. Sperm phenotypes of several of these 29 genes, including BSP5, ALOX15 and TXNDC2/SPTRX2 have already been studied in our laboratory. As expected, none of the 29 spermatogenesis/sperm related genes with LOF alleles are embryonic lethal since the functions of their products in healthy individuals are restricted to germ cells and therefore not essential for life. Among the sperm-related genes with potentially detrimental polymorphisms detected in the sequencing of 219 bulls, we have already researched two sperm proteins: 1) Spermatid-specific thioredoxin TXDNC8/SPTRX3 is retained exclusively by defective human spermatozoa. We have previouslypublished the IBFC phenotype study of SPTRX3, and established its correlation with couples' assisted reproductive therapy (ART) treatment outcomes. 2) The WBP2NL/PAWP sperm head protein contributes to sperm-induced oocyte activation during fertilization and isectopic or completely absent from spermatozoa with head malformations. Both bovine and human PAWP orthologs show potentially detrimental polymorphisms, including a C-terminal truncation caused by a premature codon (unpublished data). Our analysis of 219 sire/18 breed genome sequences identified 29 genes with high confidence LOF allelesdirectly annotated for spermatogenesis/sperm structure. Notably, a deleterious LOF allele with significant frequency was identified in the gene encoding the binder of sperm protein BSP5, resulting in a function altering Ser155-Ile substitution. We found that BSP5 is ectopically localized in spermatozoa with abnormal morphology and accumulates in the defective bull sperm fraction after semen nanopurification. The BSP family sperm surface proteins originate from seminal plasma and are important for the formation of oviductal sperm reservoir prior to fertilization. A deleterious amino acid substitution in BSP5 may significantly reduce a carrier's fertility. Additional LOF alleles in genes of interest in TABLE 1 that have been researched in our laboratory include another sperm-specific thioredoxin, TXNDC2/SPTRX2, involved together with SPTRX3 in sperm protection against oxidative stress in themouse, sperm tail protein ODF3L1 and lipoxigenase ALOX15 which we have already correlated with low fertility in boars and Alox15 -/- male mice. The innovative image-based flow cytometry (IBFC) instrument https://www.amnis.com/flowsight.html curently used in our laboratory to phenotype the above biomarkes in bull spermatozoacombines the high throughput cytometry with imaging capabilities of an epifluorescence microscope. Our group was the first to adapt IBFC for sperm analysis, and specifically for human andrology in a proof of principle study conducted in collaboration with the manufacturer, AMNIS Corporation.We have published a detailed phenotypic analysis of WBP2NL/PAWP protein using a combination of multiplex conventional FC and IBFC. This work illustrates the unsurpassed capabilities of IBFC, now available in a form of a highly accurate bench top instrument named FlowSight, eliminating the need for extrapolation between FC of sperm biomarkers and their phenotyping by light microscopy. Our aproach to identifying LOF mutations repfelcedt by bull sperm phenotypes isexemplified by new data on a proven infertile (30 infertile matings) asthenozoospermic Charolais bull with sperm tail principal piece bend similar to male-infertile c-Ros receptor KO mouse. Our sequencing of this bull revealed a deleterious homozygous-recessive mutation of the Mkrn1 gene causing abnormal accumulation of makorin 1 ubiquitin ligase in spermatozoa of this asthenozoospermic yearling, a pattern that we have also identified in defective human spermatozoa. in humans, Mkrn1 is mutated in men with polycystic kidney syndrome, and is linked to male infertility.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Dogan S, Vargovic P, Oliveira R, Belser RE, Kaya A, Moura A, Sutovsky P, Parrish J, Topper E, Memili E (2015) Sperm protamine status correlates to the fertility of breeding bulls. Biol. Reprod, 92(4): 1-9.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Sutovsky P, Aarabi M, Miranda-Vizuete A, Oko R (2015) Negative biomarker based male fertility evaluation: Sperm phenotypes associated with molecular-level anomalies. Asian J. Androl. 17, 554-60.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2016
Citation:
Oko R., Aarabi M., Mao J, Balakier H, Sutovsky P (2016) Sperm specific WW-domain binding proteins. In: The Sperm Cell: Production, Maturation, Fertilization, Regeneration. Second Edition, DeJonge C, Barrat C, Eds., Cambridge University Press, Cambridge, UK, In Press.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience:Animal sceintists, veterinarians, graduate students, cattle farmers, artificail insemination (AI) professionals Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our primary goal is to identify polymorphisms within genes that influence spermatogenesis and sperm function during fertilization. We have identified the PAWP/WBP2NL sperm head protein as a cofactor during oocyte activation. In our analysis of whole genome sequence (WGS) data on 11 Angus bulls (~20X coverage/bull), we found 9 synonymous and non-synonymous AA substitution-causing polymorphisms in PAWP, including an indel that is predicted to cause a C-terminal truncation of the PAWP protein. We conducted a flow cytometric trial in 136 Genex bulls (including 38 with CR rates from ≥100 AI services), showing a statistically significant correlation (p=0.007; r=0.399) between normal sperm PAWP content and conception rate to AI. We confirmed these findings by a detailed phenotypic analysis of PAWP in a set of 108 Alta bulls with acceptable but varied AI fertility. Based on sperm quality biomarker discovery, we have developed a method for magnetic depletion of defective spermatozoa from bull semen, prior to extender addition and preparation of AI doses/straws.We conducted two separate field trials on a total of798 Angus-Hereford cows, in both of which nanodepletion improved pregnancy rates when a half-AI-dose of 10 million sperm was compared with the control full dose of 20 million non-depleted spermatozoa. Importantly, no adverse effects on inseminated animals were observed. Both trials were conducted in collaboration with Select Sires Inc. (semen nanopurification and cryopreservation; Dr. M. De Jarnette) and USDA Research Station, Forth Keogh, MT (field AI; Dr. Tom Geary). Semen from four sires was purified with PNA- OR ubiquitin- (UBI) particles. Doses purified with UBI/PNA were used at 10 million/straw (10M), controls at 10 and 20 million. Pregnancy rates with a half dose of PNA-purified semen matched those with full control dose in both years. Nanopurified doses had no detectable side effect on inseminated animals. The UBI-nanodepleted half-dose (n=96) had a somewhat reduced pregnancy rate in year 1, compared to 10M control. This was caused by sperm overdepletion due to high particle correlation, which was improved on your 2. Also, sire-to-sire and cow vs. heifer comparisons and the significantly improved IVF fertilization rates, illustrate the potential of this treatment for improvement of AI fertility.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Odhiambo JF, DeJarnette JM, Geary TW, Kennedy CE, Suarez SS, Sutovsky M, Sutovsky P (2014). Increased Conception Rates in Beef Cattle Inseminated with Nanopurified Bull Semen. Biol. Reprod., 91(4):97.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Petruska P, Capcarova M, Sutovsky P (2014) Antioxidant supplementation and purification of semen for improved artificial insemination in livestock species. Turkish J Vet Anim Sci, 38:643-652.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kennedy CE, Krieger KB3, Sutovsky M, Xu W, Vargovi? P, Didion BA, Ellersieck MR, Hennessy ME, Verstegen J, Oko R, Sutovsky P (2014) Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination. Mol Reprod. Dev., 81(4): 436-49
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sutovsky P, Kennedy C. E. (2013) Biomarker-based nanotechnology for the improvement of reproductive performance in beef and dairy cattle. Indust. Biotech. 9: 24-30.
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